Overdose with acetaminophen (APAP) is the most common cause of acute liver failure in humans. Metabolic bioactivation of APAP initiates a cascade of events that causes hepatocellular necrosis, and it is during this progression phase when antidotal therapy is most likely to be successful. In human patients, coagulation cascade activation and thrombin generation accompanies the progression of APAP hepatotoxicity. In APAP- treated mice, tissue factor (TF) activates the coagulation cascade, resulting in activation of the thrombin receptor, protease-activated receptor-1 (PAR-1), and in the deposition of fibrin in liver. Preliminary results suggest that coagulation system activation has dichotomous roles in APAP hepatotoxicity. TF-dependent thrombin generation and stimulation of PAR-1 appear to contribute to the early progression of liver damage, since deficiency in TF or PAR-1 reduces early APAP-induced hepatocellular injury. Generation of nitric oxide and of cytokines such as tumor necrosis factor-alpha and interleukin 1-beta are associated with early progression of APAP hepatotoxicity, and PAR-1 activation of nonparenchymal cells in other tissues has been shown to stimulate production of each of these factors. Conversely, thrombin-mediated fibrin deposition limits later hepatocellular injury and hemorrhage. The deposition of fibrin is enhanced by the antifibrinolytic activity of plasminogen activator inhibitor-1 (PAI-1), a plasma protein the expression of which is induced by the transcription factor, hypoxia inducible factor-1alpha (HIF-1a). Based on our preliminary data, we hypothesize that APAP overdose results in TF-dependent production of thrombin, which contributes to the early progression of liver injury by activating PAR-1, but also limits hemorrhage and hepatocellular necrosis progression by generating fibrin. This hypothesis will be tested in mice in a series of experiments in vivo and in vitro employing parenchymal and nonparenchymal liver cells and using genetic approaches including novel mice generated using conditional knockout, Cre-LoxP technology, as well as appropriate pharmacological interventions. Aim 1 will explore the importance of TF expressed by hepatocytes and hepatocyte-derived procoagulant microparticles in thrombin generation, and the role of decreased glutathione in TF activation during APAP toxicity. Aim 2 will determine the role of PAR-1 activation on nonparenchymal cells in the expression of factors associated with the pathogenesis APAP-induced liver injury. The final aim will focus on the injury-limiting influence of fibrin clots and role of HIF-1a-mediated expression of PAI-1 in fibrin deposition in liver. Elucidating mechanisms by which APAP-induced liver injury progresses is essential for defining novel strategies to prevent liver failure in patients and for the general understanding of the pathogenesis of drug-induced liver injury. PUBLIC HEALTH RELEVANCE: Coagulation cascade activation and associated thrombin generation occur in human patients who suffer from acetaminophen overdose, a major cause of acute liver failure in the US. Studies in mice revealed that thrombin has both harmful and beneficial roles in the progression of acetaminophen-induced liver injury. The proposed studies are designed to understand the mechanisms behind these dichotomous roles, and the results could lead to improved therapy and survival for patients with acetaminophen overdose.